Abstract

Nuclear architecture has never been carefully examined during early mammalian development at the stages leading to establishment of the embryonic and extra-embryonic lineages. Heterogeneous activity of the methyltransferase CARM1 during these stages results in differential methylation of histone H3R26 to modulate establishment of these two lineages. Here we show that CARM1 accumulates in nuclear granules at the 2- to 4-cell stage transition in the mouse embryo, with the majority corresponding to paraspeckles. The paraspeckle component Neat1 and its partner p54nrb are required for CARM1's association with paraspeckles and for H3R26 methylation. Conversely, CARM1 also influences paraspeckle organization. Depletion of Neat1 or p54nrb results in arrest at the 16- to 32-cell stage, with elevated expression of transcription factor Cdx2, promoting differentiation into the extra-embryonic lineage. This developmental arrest occurs at an earlier stage than following CARM1 depletion, indicating that paraspeckles act upstream of CARM1 but also have additional earlier roles in fate choice.

CARM1 Accumulates in the Nucleus of Mouse Embryos and mESCs; Validation of Antibodies, Related to (A) Assessment of anti-CARM1 antibodies in immunofluorescense and western blots. (B) Staining to reveal CARM1 and DNA (DAPI) in the 4-cell mouse embryo with different anti-CARM1 antibodies. Scale bar 10 μm. (C) One blastomere of 2-cell stage embryo was injected with Carm1-WT synthetic mRNA and Gap43-RFP mRNA. Embryos were cultured until the 4-cell stage and subjected to immunostaining for CARM1 (#12495). (D) Confocal images of CARM1 in mESCs transfected with either control or Carm1 siRNAs. Scale bar 20 μm. (E) Carm1 knockdown reduces level of CARM1 protein in ESCs. mESCs were transfected either with control or Carm1 siRNAs for 48 h. Cell lysates were analyzed by western blotting for CARM1 and PSPC1 (loading control). (F) Differential numbers of CARM1-speckles and intensity of H3R26 immunofluorescence in single nuclei of 4-cell embryos. Nuclei of a representative embryo are shown. Scale bar 10 μm. (G) Spearman correlation coefficients of the expression of H3R26me2 and the number of CARM1 speckles (n = 99 nuclei, p < 0.0001).

CARM1 Accumulates in Nuclear Paraspeckles(A and B) Co-immunostaining of CARM1 with the paraspeckle components p54nrb and PSPC1 at the 2-cell stage (A) and 4-cell stage (B). A magnified view of single nuclei is presented at the bottom. Scale bars, 10 μm.(C) Quantification of CARM1-positive structures co-staining with p54nrb and PSPC1 (20 × 4-cell embryos, 80 nuclei in total).(D) Quantification of p54nrb and PSPC1 co-staining with CARM1-positive structures in the nucleus (20 × 4-cell embryos, 80 nuclei in total).(E) Graphical representation of a paraspeckle.

Model of the Dependency of Paraspeckle Assembly and Function on CARM1 LevelsThe assembly and structure of paraspeckles rely on constitutive transcription of its seeding lncRNA, Neat1. In the 2- and 4-cell stage embryo, Neat1 recruits paraspeckle proteins, including CARM1, to the paraspeckle structure. This sets up a negative feedback loop because of the inhibitory effect of CARM1 on Neat1 transcription. As a consequence, Neat1’s partner, p54nrb, localizes around the periphery of the nucleolus either when Neat1 is depleted or when CARM1 is overexpressed. Optimal levels of the active form of CARM1 are critical for the function and localization of p54nrb, a CARM1 substrate. Thus, we hypothesize that inhibition or loss of CARM1 results in aggregation of p54nrb by preventing its correct function as part of a positive feedback loop. Changes in the functional organization of paraspeckles through depletion of either Neat1 or p54nrb perturb the expression and function of genes involved in establishing the embryonic (ICM) and extra-embryonic (TE) lineages, including CARM1. Loss of CARM1 downstream of Neat1 and p54nrb affects the expression of pluripotency genes, as shown previously.